Long-term potentiation (LTP) is a phenomenon in which brief repetitive synaptic stimulation results in the persistent enhancement in synaptic strength. Most of our understanding of synaptic plasticity comes from studies in the CAI region of the hippocampus. At these synapses, LTP is induced by the activation of postsynaptic NMDA receptors, while the site of expression remains controversial. A less-studied form of synaptic plasticity, which has been the topic of this grant, is found at mossy fiber synapses in the CA3 region of the hippocampus. We have accomplished most of the goals laid out int he previous grant. This form of LTP is independent of NMDA receptors, and we have provided evidence that it involves entirely presynaptic mechanisms. It requires the entry of Ca into the terminal, which activates a Ca/calmodulin-sensitive adenylyl cyclase (ACI). Activation of PKA then results in the persistent enhancement of transmitter release. The goals of the present grant are seven-fold: (1) We have evidence that mossy fiber synapses activate NMDA receptors. What role might these receptors have; is there a small NMDA-sensitive component to mossy fiber LTP: Might low frequency stimulation reveal an NMDA-dependent LTD at these synapses? (2) We would predict that the presence of ACI in presynaptic terminals may be critical for the expression of mossy fiber-type LTP. We will determine if an LTP with the same properties as mossy fiber LTP can be found in the CNS by examining synapses that express ACI. Specifically, we will examine the cerebellar parallel synapses, whose cell bodies express ACI, for a mossy fiber-type, (3) We will determine the role that zinc, which is contained and released from mossy fibers, plays at these synapses. Might it normally prevent the expression of nMDA-dependent forms of plasticity by blocking these receptors: (4) If our model of LTP is correct, it should be possible to demonstrate this form of LTP at autapses in single-neuron cultures. Since all the synapses will express LTP, this reduced system will greatly facilitate studies on this form of lTP. For instance, we can determine if changes occur int he frequency or amplitude of miniature EPSCs; (5) We will apply PKA inhibitors on synapses expressing LTP to determine if the maintenance requires kinase activity; (6) We wish to develop a method to induce LTP at all mossy fiber synapses in the slice, which will permit biochemical studies on molecular mechanisms. We have preliminary evidence that high K can selectively and persistently enhance mossy fiber responses; (7) Using mouse genetics, we will determine the effects of deleting or over-expressing proteins that are candidates for involvement in mossy fiber LTP. A comparison of the results on mossy fiber LTP to those on NMDA dependent LTP will markedly advance our understanding of the basic rules underlying synaptic plasticity in the brain. Such an understanding will be indispensable in designing strategies for preventing and/or correcting the cognitive defects associated with diseases such a Alzheimer's.